fluorinated conformationally restricted γ-aminobutyric acid aminotransferase inhibitors

Fluorinated Conformationally Restricted γ-Aminobutyric Acid Aminotransferase Inhibitors

Hejun Lu and Richard B. Silverman*

Department of Chemistry, Department of Biochemistry, Molecular Biology, and Cell Biology, and the Center for Drug DiscoVery andChemical Biology, Northwestern UniVersity, EVanston, Illinois 60208-3113

ReceiVed July 24, 2006

On the basis of the structures of several potent inhibitor molecules forγ-aminobutryric acid aminotransferase(GABA-AT) that were previously reported, six modified fluorine-containing conformationally restrictedanalogues were designed, synthesized, and tested as GABA-AT inhibitors. The syntheses of all six moleculesfollowed from a readily synthesized ketone intermediate. Three of the molecules were found to be irreversibleinhibitors of GABA-AT with comparable or largerkinact/KI values than that of vigabatrin, a clinically usedantiepilepsy drug, and the other three were reversible inhibitors. A possible mechanism for inactivation byone of the inactivators is proposed.

There are two principal neurotransmitters involved in theregulation of brain neuronal activity:γ-aminobutyric acid(GABAa), one of the most widely distributed inhibitory neu-rotransmitters, andL-glutamic acid, an excitatory neurotrans-mitter.1 The GABA concentration is regulated by two pyridoxal5′-phosphate (PLP)-dependent enzymes,L-glutamic acid decar-boxylase (GAD), which catalyzes the conversion ofL-glutamateto GABA, and GABA aminotransferase (GABA-AT), whichdegrades GABA to succinic semialdehyde (SSA) concomitantwith the conversion ofR-ketoglutarate toL-glutamate.2 Whenthe concentration of GABA diminishes below a threshold levelin the brain, convulsions result;3 raising the brain GABA levelsterminates the seizure.4 A reduction in the concentrations ofGABA and of the enzyme GAD has been implicated not onlyin the symptoms associated with epilepsy5,6 but also with severalother neurological diseases such as Huntington’s chorea,7,8

Parkinson’s disease,9,10 Alzheimer’s disease,11 and tardivedyskinesia.12 GABA brain levels cannot be increased by admini-stration of GABA because it does not cross the blood-brainbarrier. An approach that has been successful to increase brainGABA levels is the use of a compound that crosses the blood-brain barrier and then inhibits or inactivates GABA-AT, therebyincreasing the GABA concentration. Numerous competitiveinhibitors of GABA-AT, particularly compounds having asimilar backbone structure to GABA,13 show anticonvulsantactivity. A variety of mechanism-based inactivators14 ofGABA-AT15 also have been shown to be effective anticonvul-sant agents. The most effective of the mechanism-basedinactivators as an anticonvulsant agent is 4-amino-5-hexenoicacid (1, Figure 1;γ-vinyl-GABA),16 which has the commercialgeneric name vigabatrin. This compound, which has highpotency,17 has been shown to be an effective treatment forepilepsies that are resistant to other anticonvulsant drugs18 andcurrently is prescribed in over 60 countries worldwide, but itwas not approved in the U.S. It also was found that vigabatrin

prevents cocaine addiction in rats and baboons.19 This activitywas observed with other forms of addiction, including nicotineaddiction20 and methamphetamine, alcohol, and heroin addic-tions.21 Self-administration of cocaine by rats decreased or wasprevented by vigabatrin administration in a dose-dependentfashion, without affecting the craving for food.22 GABA wasfound to antagonize the extracellular dopamine levels respon-sible for drug addiction, and by positron emission tomography(PET) in primates, it was shown that vigabatrin, which causesa rise in GABA levels, inhibits these drug-induced dopamineincreases.21 However, because vigabatrin is not approved in theU.S., new GABA-AT inhibitors and inactivators are needed.

Conformationally restricted analogues of GABA are ofconsiderable interest among medicinal chemists.23 We previ-ously reported the design and synthesis of a series of confor-mationally restricted analogues of GABA. Among them,(1R,4S)-4-aminocyclopent-2-enecarboxylic acid (2) was shownto be a potent inhibitor and substrate of GABA-AT, but it wasnot a time-dependent inactivator.24 (1S,3S)-3-Amino-4-methyl-enecyclopentanecarboxylic acid (3) was found to inactivateGABA-AT but only in the absence of 2-mercaptoethanol (indi-cating that a reactive species was produced that was releasedfrom the enzyme prior to inactivation), while (1S,3S)-3-amino-4-difluoromethylenecyclopentanecarboxylic acid (4) is a muchmore potent time-dependent inhibitor ofγ-aminobutyric acidaminotransferase, even in the presence of 2-mercaptoethanol(indicating that any reactive species generated are not releasedprior to inactivation).25 Compound4 is 187 times more potentof an inactivator of GABA-AT than vigabatrin, and thecorrespondingE- and Z-monofluoro analogues of3 alsoinactivate GABA-AT but not with the same potency as4. Thissuggests an important significance for fluorine in the molecules.

Fluorine and fluoroalkyl substitutents have an importantelectronic effect on the neighboring groups in a molecule andalso mimic hydrogen and alkyl groups, respectively.26 Therefore,the introduction of one or more fluorine-containing groups intomolecules alters their physical properties, as well as theirbiological activities.27 Fluorinated substrates also can be usedas mechanistic probes and inhibitors for obtaining informationabout the catalytic mechanism of various enzymatic transforma-tions.28 Consequently, it was thought that other fluorinatedconformationally restricted analogues of2 and 3 should beevaluated for their inhibitory activities. On the basis of thathypothesis, we designed six new fluorine-containing confor-mationally restricted vigabatrin analogues (5-10, Figure 2).

* To whom correspondence should be addressed. Address: Departmentof Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, IL60208-3113. Phone: 847-491-5653. Fax: 847-491-7713. E-mail: [email protected].

a Abbreviations: CAN, ceric ammonium nitrate; DBDMH, 1,3-dibromo-5,5-dimethylhydantoin; GABA,γ-aminobutyric acid; GABA-AT,γ-ami-nobutyric acid aminotransferase; GAD,L-glutamic acid decarboxylase;LiHMDS, lithium hexamethyldisilazide; MFSDA, methyl fluorosulfonyldi-fluoroacetate; PMBCl,p-methoxybenzyl chloride; SSA, succinic semial-dehyde dehydrogenase; TBAF, tetrabutylammonium fluoride, TPAP, tetra-n-propylammonium perruthenate.

7404 J. Med. Chem.2006,49, 7404-7412

10.1021/jm0608715 CCC: $33.50 © 2006 American Chemical SocietyPublished on Web 11/10/2006

Compounds5-7 are C-3 fluorine or fluoroalkyl substitutedanalogues of2, while 8-10 are trifluoromethylated analoguesof 3 and 4. The presence of strongly electron-withdrawingfluorine and fluoroalkyl groups also may enhance bindingand/or reactivity of these compounds during GABA-AT turnoverbased on a Michael addition mechanism.23a The results of thisinvestigation are reported here.

Results and Discussion

Chemistry. Key intermediate16 for the synthesis of all ofthe analogues was synthesized according to a modification of aprocedure we reported previously29 (Scheme 1). Protection of11 was realized by treatment withp-methoxybenzyl chloride(PMBCl) in the presence of sodium hydride in DMF instead ofLiHMDS in THF as reported. This revised procedure turnedout to be much easier and gave higher yields of product.

The synthesis of5 was first attempted by a direct halogenexchange reaction of17. However, treatment of17 with(PhSO2)2NF/nBuLi resulted in theent-12 reduction product asthe major product. Conversion of17 to organotin intermediate18 followed by fluorine-metal exchange with XeF2 afforded19 in a satisfactory yield (Scheme 2). Deprotection of19 withceric ammonium nitrate (CAN) followed by acidic hydrolysisgave5.

Syntheses of target molecules6 and7 via elimination of analcohol or the corresponding tosylated derivative were unsuc-cessful (Scheme 3). Treatment of16 with TMSRf (Rf ) CF3

or CF3CF2) in the presence of a catalytic amount of TBAFafforded tertiary alcohols21a and 21b in excellent yields.30

However, direct dehydration of21a or 21b with variousdehydration conditions failed to produce the desired products22a and 22b. Activation of 21a and 21b with TsCl in the

presence of NaH successfully gave the tosylates23a and23b,respectively. Unfortunately, attempted elimination reactions of23a and23b with various bases always resulted in formationof 21a and21b, respectively, with only a trace amount of thedesired products detected.

An alternative synthesis of target molecule6 (Scheme 4)started with iodination of16with hydrazine and iodine followedby elimination of one molecule of HI in the presence ofpotassiumtert-butoxide to give17. Direct trifluoromethylationof 17 with in situ generated CF3Cu from FSO2CF2CO2Me(methyl fluorosulfonyldifluoroacetate, MFSDA) and CuI31 suc-cessfully afforded22a. Removal of the PMB group with CANfollowed by acidic hydrolysis gave6.

Compound7 was synthesized from17 using steps similar tothose used to prepare6 (Scheme 5). A first attempt atpentafluoroethylation of17 with CF3CF2CO2Na/CuI at 140°Conly resulted in decomposition of the substrate. Treatment of17 with CF3CF2SiMe3/KF/CuI, however, afforded22b in goodyields.

A Wittig reaction of16 with CHBrdPPh3, generated in situfrom bromomethyltriphenylphosphonium bromide andtBuOKafforded anE/Z mixture of bromomethylenes25a and 25b,which was easily separated by column chromatography on silicagel. The conformations of the double bonds in the two isomerswere determined based on NOE experiments. Trifluorometh-ylation of 25a and 25b with CF3Cu under similar conditionsused for17 produced compounds26a and 26b, respectively.Removal of the PMB protecting group with CAN followed byacidic hydrolysis gave8 and9 (Scheme 6).

Treatment of16 with CBr4 and PPh3 in toluene afforded28,which was double-trifluoromethylated with in situ generatedCF3Cu to give 29. Removal of the PMB group with CAN,followed by hydrolysis with 4 N HCl(aq) at 75°C gave10(Scheme 7).

It is noteworthy that, compared to the compounds with anexocyclic double bond (27a, 27b, and 30), the hydrolysis of20, 24a, and24b, which have endocyclic double bonds, wasfound to be much easier. The reaction is usually completed in1 h when the compounds are treated with 4 N aqueous HCl at70 °C. Prolonged heating and stirring of these compounds underthese conditions resulted in major side reactions. No such sidereactions were observed from hydrolysis of27a, 27b, and30.

Enzyme Inhibition Results.Compounds6, 8, and9 showedconcentration and time-dependent inhibition of pig brainGABA-AT in the presence ofâ-mercaptoethanol (Table 1).Compounds5, 7, and10showed only weak reversible inhibitionof GABA-AT in the presence ofâ-mercaptoethanol. None ofthe three reversible inhibitor target molecules was more potentthan 2 or 4. However, the irreversible inhibitors were com-parable to vigabatrin as inactivators of GABA-AT. It isinteresting that although6 was designed to be a reversibleinhibitor of GABA-AT because a simple elimination of HF wasnot initially apparent, it was found to be an irreversible inhibitor.Compounds5 and7, which differ from6 only by the length ofthe fluoroalkyl chain, are reversible inhibitors of GABA-AT.Although 8 and9 are irreversible inhibitors of GABA-AT, asexpected, introduction of a second trifluoromethyl group makes10 a weak reversible inhibitor.

Figure 1. Vigabatrin (1) and conformationally restricted analogues.

Figure 2. New fluorinated conformationally restricted potentialinhibitors and inactivators of GABA-AT.

Scheme 1a

a Reagents and conditions: (a) PMBCl, NaH, Bu4NI, DMF, 0 °C to roomtemp, 2 h, 83%; (b) DBDMH, HOAc, room temp, 20 h, 97%; (c) Bu3SnH,AIBN, PhH, reflux, 12 h, 95%; (d) K2CO3, MeOH, H2O, room temp, 2 h,87%; (e) NMO, TPAP, DCM, room temp, 24 h, 70%.

γ-Aminobutyric Acid Aminotransferase Inhibitors Journal of Medicinal Chemistry, 2006, Vol. 49, No. 257405

Exhaustive dialysis of GABA-AT that was inactivated by6,8, and 9 against potassium pyrophosphate buffer (pH 8.5)resulted in different extents of irreversibility of inhibition.Compared to the control, 48% and 13% of enzyme activity wasrecovered for6 and8, respectively, after 22 h of dialysis, whilethe percentage of recovered enzyme activity for9 continued toincrease with extended dialysis. These results suggest that atleast part of the adducts formed between the enzyme and6 and8 are stable, while at least one with9 can be slowly hydrolyzedupon dialysis. Incubation of6, 8, and9 with GABA-AT in thepresence of GABA resulted in a significant increase in theirt1/2 of inactivation (slower rate of inactivation), indicating active-site binding to GABA-AT by these compounds.32

A possible mechanism for inactivation of GABA-AT by6 isshown in Scheme 8. Initial Schiff base formation with the PLPfollowed by tautomerization gives31. Michael addition to thistrifluoromethyl-activated Michael acceptor33 by Lys329 (X)

Lys329) or hydroxide (X) HO-) results in32, which caneliminate fluoride ion to give a highly reactive exocyclicdifluoromethylene group32 conjugated to the PMP iminium (33).This should lead to rapid nucleophilic attack,32 either by theenzyme (pathway a) or by water (pathway b) to give34 or 36,respectively. Fluoride ion elimination and hydrolysis would give35 (inactivation) or38 (turnover). Inactivation by8 and9 maybe related to the mechanism of inactivation proposed for4.25

To test this mechanism, fluoride ion release was monitoredduring inactivation. GABA-AT was incubated with all sixcompounds for 2 h, and the fluoride ion concentration wasmonitored. With6, 270 fluoride ions were released per enzyme

Scheme 2a

a Reagents and conditions: (a) NH2NH2‚H2O, Et3N, EtOH, reflux, 1 h; (b) I2, Et3N, benzene, room temp, 2 h; (c)tBuOK, ether, room temp, 20 h, 60%in three steps; (d) Me3SnSnMe3, Pd(PPh3)4, PhMe, reflux, 30 min, 49%; (e) XeF2, AgOTf, 2,6-bis(tert-butyl)-4-methylpyridine, DCM, room temp, 8 min,30%; (f) CAN, CH3CN, H2O, room temp, 2 h, 55%; (g) 4 N HCl(aq), 70°C, 0.5-1 h, 64%.

Scheme 3a

a Reagents and conditions: (a) TMSRf, TBAF(cat.), THF, room temp,1 h, 95%; (b) TsCl, NaH, ether, 0°C, 16 h, 79%.

Scheme 4a

a Reagents and conditions: (a) MFSDA, CuI, DMF, HMPA, 20 h, 75%;(b) CNA, MeCN/H2O, room temp, 3 h, 61%; (c) 4 N HCl(aq), 70°C, 0.5-1h, 85%.

Scheme 5a

a Reagents and conditions: (a) CF3CF2SiMe3/KF/CuI, NMO/DMF(1/1), 75°C, 24 h, 57%; (b) CAN, CH3CN, H2O, room temp, 2 h, 76%; (c)4 N HCl(aq), 70°C, 0.5-1 h, 82%.

Scheme 6a

a Reagents and conditions: (a) BrCH2PPh3.Br, tBuOK, THF, -78 °C,20 h, 72%E/Z; (b) MFSDA, CuI, DMF, HMPA, 75°C, 20 h; 95%; (c)CAN, CH3CN, H2O, room temp, 2 h, 76%; (d) 4 N HCl(aq), 70°C, 10-12h, 79%.

Scheme 7a

a Reagents and conditions: (a) CBr4, PPh3, toluene, reflux 22 h, 86%;(b) MFSDA, CuI, DMF, HMPA, 75°C, 50 h, 82%; (c) CAN, CH3CN,H2O, room temp, 1 h, 56%; (d) 4 N HCl(aq), 70°C, 10-12 h, 77%.

Table 1. Kinetic Constants for5-10

compdkinact

(min-1)KI

(mM)kinact/KI

(min-1 mM-1)Ki

(mM)

5 1.36 0.68 4.11 0.177 2.88 6.96 39.23 0.189 2.89 7.74 0.3710 4.2vigabatrina 0.24 0.85 0.28

a From ref 25.

7406 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 25 Lu and SilVerman

dimer inactivated. This suggests that possibly 90 molecules of6 are turned over for each inactivation event (partition ratio of89). Possibly, the activated intermediate33 undergoes reactionwith water (pathway b) 89 times for each attack by an activesite residue (pathway a). Depending on what enzyme residuereacts with33, the carboxylic acid derivative product formedmay or may not be stable to conditions of dialysis, leading topartial reactivation of the enzyme during dialysis. For7, 109fluoride ions were released, and for the other four compounds,less than 6 fluoride ions were released during inactivation.

Enzyme activity was monitored concomitantly with fluorideion release during inactivation of GABA-AT by6. A goodcorrelation was observed (Figure 3), which suggests thatinactivation occurs as a consequence of fluoride ion release.When GABA-AT was substituted by human albumin, nofluoride ion was released, indicating that the presence of aprotein is not responsible for the fluoride ion release.

Figure 4 shows the time-dependent increase in fluoride ionconcentration when GABA-AT was incubated with7, a weakreversible inhibitor. The fluoride ion concentration reached ithighest level in 2-3 h. No fluoride ion was released when7was incubated with albumin, indicating a GABA-AT-dependentreaction. This suggests that the corresponding reactive inter-mediate (related to33 in Scheme 8) only undergoes hydrolysis(possibly because of steric hindrance by the trifluoromethylgroup of7 that replaces the fluoride in6).

Conclusion

Two series of fluorinated compounds were designed aspotential inhibitors and inactivators of GABA-AT based on thehigh potency of4. Thekinact/KI value for8 (0.37 mM-1 min-1)is greater than that for the epilepsy drug vigabatrin (0.28 mM-1

min-1); that for 6 (0.16 mM-1 min-1) and for9 (0.18 mM-1

min-1) are slightly lower. None of these, however, is as effi-cient an inactivator as4 (52 mM-1 min-1). Compounds5-7were designed as reversible inhibitors, so it was unexpected that

Scheme 8

Figure 3. Enzyme activity and fluoride ion release during inactivationof GABA-AT with 6 (2 mM) as a function of time: fluoride ionconcentration when GABA-AT was incubated with6 (diamonds);fluoride ion concentration when human albumin was incubated with6(squares); remaining relative enzyme activity compared to control whenGABA-AT was incubated with6 (triangles).

Figure 4. Enzyme activity and fluoride ion release during inac-tivation of GABA-AT with 7 (2 mM) as a function of time: fluorideion concentration when GABA-AT was incubated with7 (diamonds);remaining relative enzyme activity compared to control whenGABA-AT was incubated with7 (squares).


6 was a potent time-dependent inactivator. It appears that thereis more to the effective binding of compounds to GABA-ATthan just the fluorine atoms. Other structures that more closelyresemble4 are being constructed.

Experimental Section

General Methods and Reagents. Optical spectra andGABA-AT assays were recorded on a Perkin-Elmer Lambda 10UV/vis spectrophotometer.1H NMR spectra were recorded on aVarian Gemini 300 MHz NMR spectrometer. Chemical shifts arereported asδ values in parts per million downfield from TMS asthe internal standard in CDCl3. For samples run in D2O, the HODresonance was arbitrarily set at 4.60 ppm. CClF3 was selected asan external standard withδ 0.0 ppm for19F NMR. Melting pointswere determined on a Fisher-Johns melting point apparatus andare uncorrected. Combustion analyses were performed by OneidaResearch Laboratories, NY. High-resolution mass spectra andaccurate mass spectra were obtained on a VG-70-250SE high-resolution spectrometer and a Micromass Quattro II LC/MSspectrometer. An Orion Research model 702A pH meter with ageneral combination electrode was used for pH measurements.Fluoride ion concentration measurements were obtained using anOrion Research model 702A pH meter with an Orion Researchmodel 96-09 combination fluoride electrode. Flash column chro-matography was carried out with Merck silica gel 60 (230-400mesh ASTM). TLC was run with EM Science silica gel 60 F254precoated glass plates.

All reagents were purchased from Aldrich Chemical Co. andwere used without further purification except anhydrous ether andtetrahydrofuran (THF), which were distilled from sodium metalunder nitrogen and anhydrous dichloromethane, which was distilledfrom calcium hydride.

(1S,4R)-6-Iodo-2-(4-methoxybenzyl)-2-azabicyclo[2.2.1]hept-5-en-3-one (17).Compound16 (0.246 g, 1.0 mmol)29 was dissolvedin anhydrous ethanol (5 mL) and was added via syringe to a solutionof hydrazine hydrate (51% of hydrazine, 1.02 mL, 21.0 mmol) andEt3N (2.18 mL, 15.7 mmol) in anhydrous ethanol (5 mL) whilestirring. The resulting colorless solution was heated to reflux andstirred under argon for 1 h. The reaction mixture was thenevaporated under vacuum to give the crude hydrazone product asa colorless oil, which was used directly in the next step withoutfurther purification. I2 (0.508 g, 2.0 mmol) was dissolved inanhydrous benzene (6 mL) and was added dropwise into a solutionof the above hydrazone and Et3N (1.1 mL, 8.0 mmol) in anhydrousbenzene (5 mL) with stirring at room temperature. The resultingbrown suspension was stirred at room temperature under argon for1.5 h. Water (20 mL) was added, and the resulting two layers wereseparated; the aqueous layer was further extracted with ether (20mL × 3). The combined organic layers were washed with aqueousHCl (0.5 N, 10 mL), water (10 mL), saturated aqueous NaHCO3

(20 mL), and brine (20 mL× 2) and dried over MgSO4. The solventwas removed under vacuum, and the resulting crude product waspurified via column chromatography on silica gel (hexanes/EtOAc,9:1) to give the diiodo intermediate as a light-brown oil, whichwas dissolved in anhydrous ether and treated by dropwise additionof two equiv of tBuOK at room temperature; the resulting brownsuspension was stirred overnight. Water was added, and the twolayers were separated. The aqueous layer was further extracted withether, and the combined ether solutions were washed with brineand dried over anhydrous MgSO4. Ether was removed undervacuum, and the resulting crude product was purified via columnchromatography on silica gel (hexanes/EtOAc, 9:1) to give17 asa light-green oil (213 mg, 60%).1H NMR (400 MHz, CDCl3) δ7.19 (2H, d,J ) 8.5 Hz), 6.96 (1H, s), 6.89 (2H, d,J ) 8.5 Hz),4.53 (1H, d,J ) 14.5 Hz), 4.06 (1H, s), 4.01 (1H, d,J ) 14.5 Hz),3.82 (3H, s), 3.36 (1H, s), 2.31 (1H, d,J ) 7.5 Hz), 2.22 (1H, d,J ) 7.5 Hz). 13C NMR (100 MHz, CDCl3) δ 178.50, 159.36,144.04, 129.84, 128.73, 114.32, 98.91, 71.65, 58.13, 56.33, 55.38,46.83. MSm/z (ESI, MeOH): 378 (M+ Na)+. HRMS calcd forC14H14O2NI: 355.0064. Found: 355.0057.

(1S,4R)-2-(4-Methoxybenzyl)-6-trimethylstannanyl-2-azabicyclo-[2.2.1]hept-5-en-3-one (18).Compound17 (41 mg, 0.115 mmol)was dissolved in anhydrous toluene (4 mL) under argon, followedby addition of Pd(PPh3)4 (20 mg, 0.017 mmol) and Me3SnSnMe3(45 mg, 0.139 mmol). A light-yellow solution formed, which washeated to reflux under argon and stirred for 1 h. Toluene wasevaporated under vacuum, and the residue was purified via columnchromatography on silica gel (hexanes/EtOAc, 3:1) to give18 (25mg, 56%) as a colorless oil.1H NMR (500 MHz, CDCl3) δ 7.14(2H, d,J ) 8.5 Hz), 6.88 (2H, d,J ) 8.5 Hz), 6.85 (1H, m), 4.67(1H, d,J ) 15.0 Hz), 4.13 (1H, s), 3.81 (3H, s), 3.47 (1H, s), 3.41(1H, d, J ) 15.0 Hz), 2.24 (1H, d,J ) 7.5 Hz), 2.00 (1H, d,J )8.0 Hz), 0.16 (9H, s).13C NMR (CDCl3, 125.6 MHz)δ 179.83,159.03, 153.07, 148.19, 129.26, 129.12, 114.12, 66.20, 58.27, 55.68,55.34, 47.56,-9.28. MSm/z (EI, DCM): 389.0 (M)+, 374.0 (M- CH3)+, 211.0 (M- PMB - CONH - CH3)+, 121.0 (PMB)+.HRMS calcd for C17H23O2N116Sn (M)+: 389.0741. Found: 389.0749.HRMS calcd for C16H20O2N116Sn (M - CH3)+: 374.0506. Found:374.0504.

(1S,4R)-6-Fluoro-2-(4-methoxybenzyl)-2-azabicyclo[2.2.1]hept-5-en-3-one (19).AgOTf (48 mg, 0.187 mmol) was dissolved indry DCM under argon. Compound18 (73.4 mg, 0.187 mmol) and2,6-di-tert-butyl-4-methylpyridine (19 mg, 0.094 mmol) in dryDCM (1 mL) were added via syringe, followed by immediateaddition of XeF2 (38 mg, 0.225 mmol) in DCM. The resultingreaction mixture was shielded from light and stirred at roomtemperature for 15 min. The reaction mixture was then partitionedbetween saturated aqueous NaHCO3 and CHCl3, and the aqueouslayer was further extracted with CHCl3 (20 mL). The combinedCHCl3 solutions were washed with brine (20 mL) and dried overanhydrous MgSO4. The CHCl3 was removed under vacuum, andthe residue was purified via column chromatography on silica gel(hexanes/EtOAc, 3:1) to give19 (20 mg, 30%) as a light-yellowoil. 1H NMR (400 MHz, CDCl3) δ 7.15 (2H, d,J ) 8.8 Hz), 6.86(2H, d,J ) 8.4 Hz), 5.57 (1H, s), 4.48 (1H, d,J ) 14.8 Hz), 4.08(1H, d,J ) 14.8 Hz), 3.89 (1H, s), 3.80 (1H, s), 3.24 (1H, s), 2.33- 2.38 (2H, m).19F NMR (376 MHz, CDCl3) δ -119.72 (1F, s).13C NMR (CDCl3, 100.63 MHz)δ 177.01, 173.97, 159.20, 129.68,128.27, 114.11, 105.37 (d,J ) 7.3 Hz), 62.64, 57.16, 55.33, 49.62,47.12. MSm/z (EI, DCM): 247.1 (M)+, 163.1, 121.1 (PMB)+.HRMS calcd for C14H14O2NF (M)+: 247.1003. Found: 247.1003.

(1S,4R)-6-Fluoro-2-azabicyclo[2.2.1]hept-5-en-3-one (20).Thiscompound was synthesized from19using general procedure II (seebelow) in a 55% yield.1H NMR (400.168 MHz, CDCl3) δ 6.41(1H, s), 5.59 (1H, s), 4.12 (1H, s), 3.10 (1H, s), 2.44 (2H, s).19FNMR (376.492 MHz, CDCl3) δ -120.59 (1F, s).13C NMR (CDCl3,125.614 MHz)δ 184.95, 175.63 (d,JFC ) 304.9 Hz), 105.88, 59.52,58.49, 49.45. MSm/z (CI, DCM): 128.1 (M + 1)+, 85.3 (M -CON)+. HRMS calcd for C6H7ONF: 128.0506. Found: 128.0511.

(1R,4S)-4-Amino-3-fluorocyclopent-2-enecarboxylic Acid (5).This compound was synthesized from20 using general procedureIII in a yield of 64% as a light-yellow solid, mp 201.0-202.5°C.1H NMR (D2O, 499.511 MHz)δ 5.68 (1H, s), 4.45 (1H, d,J )2.9 Hz), 3.66 (1H, d,J ) 4.0 Hz), 2.83 (1H, dt,J ) 14.5 Hz, 9.0Hz), 2.20 (1H, dt,J ) 14.5 Hz, 4.5 Hz).19F NMR (D2O, 376.493MHz) δ -128.97 (1F, d,J ) 4.5 Hz). 13C NMR (D2O, 125.614MHz) δ 177.18, 157.61 (d,JFC ) 280.2 Hz), 109.21 (d,JFC )12.1 Hz), 51.37 (d,JFC ) 22.2 Hz), 42.57 (d,JFC ) 8.2 Hz), 29.32(d, JFC ) 4.0 Hz). MSm/z (EI, MeOH): 145.1 (M)+, 126.1, 100.1(M - CO2H)+. HRMS calcd for C6H8O2NF: 145.0534. Found:145.0530. Anal. (C6H8O2NF‚0.5H2O) C, H, N.

(1S,4S,6SR)-6-Hydroxy-6-trifluoromethyl-2-(4 ′-methoxybenzyl)-azabicyclo[2.2.1]heptan-3-one (21a).To a solution of16 (0.123g, 0.5 mmol) in dry THF (4 mL) was added TMSCF3 (0.107 mg,0.75 mmol). Tetrabutylammonium fluoride (TBAF) (1.0 M solutionin THF, 50 µL, 0.05 mmol) was added dropwise. The resultingmixture was stirred under nitrogen for 2 h and was concentratedunder vacuum. The residue was purified by column chromatography(hexanes/EtOAc, 2:1 to 1:1), giving21a(0.149 g, 94%) as a whitesolid. 1H NMR (400 MHz, CDCl3) δ 7.19 (2H, d,J ) 8.4 Hz),6.87 (2H, d,J ) 8.4 Hz), 5.00 (1H, d,J ) 14.8 Hz), 4.56 (1H, br.


s), 4.04 (1H, d,J ) 14.4 Hz), 3.81 (3H, s), 3.74 (1H, s), 2.87 (1H,s), 2.33 (1H, dd,J ) 14.0 Hz, 3.6 Hz), 1.80-1.91 (3H, m).19FNMR (376 MHz, CDCl3) δ -78.19 (3F, s). MSm/z (EI, DCM):316.1 (M + 1)+, 315.1 (M)+, 121.1 (CH3OC6H4CH2)+. HRMScalcd for C15H16O3NF3: 315.1070. Found: 315.1077.

(1S,4S,6SR)-6-Hydroxyl-6-pentafluoroethyl-2-(4′-methoxy-benzyl)azabicyclo[2.2.1]heptan-3-one (21b).This compound wassynthesized by the same method as for21a. 1H NMR (400 MHz,CDCl3) δ 7.17 (2H, d,J ) 8.8 Hz), 6.87 (2H, d,J ) 7.6 Hz), 4.98(1H, d,J ) 15.2 Hz), 4.97 (1H, s), 4.01 (1H, d,J ) 14.8 Hz), 3.86(1H, s), 3.80 (3H, s), 2.84 (1H, s), 2.42 (1H, d,J ) 13.6 Hz),1.78-1.89 (3H, m).19F NMR (376 MHz, CDCl3) δ -79.02 (3F,s), -120.08 (2F, ab,J ) 277.5 Hz). MSm/z (EI, DCM): 366.1(M + 1)+, 365.1 (M)+, 121.1 (CH3OC6H4CH2)+. HRMS calcd forC16H16O3NF5: 365.1050. Found: 365.1045.

(1S,4R,6S)-Toluene-4-sulfonic Acid 2-(4-Methoxybenzyl)-3-oxo-6-trifluoromethyl-2-azabicyclo[2.2.1]hept-6-yl Ester (23a).NaH in mineral oil (14 mg, 60%, 0.3 mmol) was washed withanhydrous pentane, suspended in dry ether (3 mL), and cooled to0 °C in an ice bath. Compound21a (52 mg, 0.16 mmol) in dryether (1 mL) was then added dropwise via syringe. The resultingwhite suspension was stirred at 0°C for 15 min under argonfollowed by addition of TsCl (64 mg, 0.3 mmol) in dry ether (1mL). The resulting white suspension was allowed to slowly warmup to room temperature and was stirred under argon for 16 h. Waterwas added, and the two layers were separated. The aqueous layerwas further extracted with ether (10 mL× 3), and the combinedether solutions were washed with brine (10 mL× 2) andconcentrated under vacuum. The residue was separated by flashcolumn chromatography on silica gel (hexanes/EtOAc, 1:1) to give23a (58 mg, 79%) as a white solid.1H NMR (500 MHz, CDCl3)δ 7.82 (2H, d,J ) 8.0 Hz), 7.40 (2H, d,J ) 6.0 Hz), 7.04 (2H, d,J ) 6.4 Hz), 6.82 (2H, d,J ) 6.4 Hz), 4.75 (1H, d,J ) 12.0 Hz),3.84 (1H, s), 3.78 (3H, s), 3.43 (1H, d,J ) 12.0 Hz), 3.09 (1H, d,J ) 12.0 Hz), 2.96 (1H, s), 2.47 (3H, s), 2.40 (1H, dd,J ) 12.4Hz, 2.8 Hz), 1.81-1.84 (2H, m).19F NMR (376 MHz, CDCl3) δ-74.41 (3F, s).13C NMR (CDCl3, 125.6 MHz)δ 175.17, 159.24,145.72, 134.22, 130.13, 129.54, 128.30, 127.76, 123.16 (q), 114.21,63.32, 55.31, 45.83, 45.03, 39.13, 31.11, 21.80.

(1S,4R,6S)-Toluene-4-sulfonic Acid 2-(4-Methoxybenzyl)-3-oxo-6-pentafluoroethyl-2-azabicyclo[2.2.1]hept-6-yl Ester (23b).This compound was synthesized from21b similar to the synthesisof 23a. 1H NMR (400 MHz, CDCl3) δ 7.84 (2H, d,J ) 8.0 Hz),7.40 (2H, d,J ) 8.8 Hz), 7.04 (2H, d,J ) 8.4 Hz), 6.83 (2H, d,J ) 8.0 Hz), 4.95 (1H, d,J ) 15.6 Hz), 3.90 (1H, s), 3.78 (3H, s),3.54 (1H, d,J ) 14.8 Hz), 3.29 (1H, d,J ) 15.6 Hz), 2.96 (1H, s),2.48-2.51 (4H, m), 1.85 (1H, d,J ) 11.2 Hz), 1.79 (1H, d,J ) 10.4 Hz). 19F NMR (376 MHz, CDCl3) δ -78.99 (3F, s),-116.41 (2F, ab).13C NMR (CDCl3, 125.6 MHz)δ 174.98, 159.22,145.86, 133.85, 130.04, 129.62, 127.98, 114.21, 63.21, 55.33,46.13, 44.59, 30.77, 21.87. MSm/z (EI, MeOH): 519.0 (M)+, 520.0(M + 1)+, 365 (M - CH3C6H4SO2)+, 121.0. HRMS calcd forC23H22O5NF5S: 519.183. Found: 519.1135.

(1S,4R)-6-Trifluoromethyl-2-(4-methoxybenzyl)-2-azabicyclo-[2.2.1]hept-5-en-3-one (22a). General Procedure I.Methyl fluo-rosulfonyldifluoroacetate (MFSDA; 48 mg, 0.25 mmol) in anhy-drous DMF (1 mL) was added dropwise via syringe to a suspensionof 17 (35.5 mg, 0.1 mmol) and CuI (23 mg, 0.12 mmol) inanhydrous DMF (2 mL) and HMPA (1 mL) at 75°C under argonover a period of 1 h. The resulting suspension was stirred at 75°Cunder argon for 72 h and evaporated under high vacuum. Theresidue was purified via column chromatography on silica gel(hexanes/EtOAc, 3:1) to give22a(22.1 mg, 75%) as a light-brownoil. 1H NMR (500 MHz, CDCl3) δ 7.18 (2H, d,J ) 8.5 Hz), 7.11(1H, s), 6.89 (2H, d,J ) 8.5 Hz), 4.77 (1H, d,J ) 15.0 Hz), 4.23(1H, s), 3.82 (3H, s), 3.56 (1H, s), 3.54 (1H, d,J ) 15.0 Hz), 2.40(1H, d, J ) 8.0 Hz), 2.32 (1H, d,J ) 8.0 Hz).19F NMR (376.49MHz, CDCl3) δ -64.69 (3F, s).13C NMR (CDCl3, 125.6 MHz)δ178.04, 159.30, 143.97 (q,JCF ) 36.6 Hz), 141.52 (q,JCF ) 5.4Hz), 129.54, 128.26, 122.44 (q,JCF ) 267.8 Hz), 114.25, 61.35,59.31, 55.35, 54.06, 46.81. MSm/z (EI, MeOH): 297.1 (M)+, 298.1

(M + 1)+, 163.1 (M- PMBN)+, 121.0 (PMB)+. HRMS calcd forC15H14O2NF3: 297.0971. Found: 297.0970.

(1S,4R)-6-Trifluoromethyl-2-azabicyclo[2.2.1]hept-5-en-3-one (24a). General Procedure II.Ceric ammonium nitrate (CAN;1.38 g, 2.525 mmol) in water (3 mL) was added to a solution of22a (150 mg, 0.505 mmol) in acetonitrile (6 mL). The resultingred solution was stirred at room temperature for 1.5 h. The reactionmixture was diluted with ethyl acetate, washed with saturatedaqueous NaHCO3 and then brine, and dried over anhydrous MgSO4.The solution was concentrated under vacuum, and the residue waspurified via column chromatography on silica gel (hexanes/EtOAc,1:1) to give24a (54 mg, 61%) as a white solid.1H NMR (500MHz, CDCl3) δ 7.07 (1H, s), 6.15 (1H, br. s), 4.51 (1H, s), 3.42(1H, s), 2.54 (1H, d,J ) 8.0 Hz), 2.43 (1H, d,J ) 8.0 Hz). 19FNMR (376 MHz, CDCl3) δ -66.42 (3F, s).13C NMR (CDCl3, 125.6MHz) δ 182.3, 144.3 (m), 141.0 (q,JFC ) 5.5 Hz), 122.4 (q,JFC

) 268.2 Hz), 60.1, 58.6, 53.7. MSm/z (CI, MeOH): 178.3 (M+1)+, 115.2. HRMS calcd for C7H7ONF3 (M + H): 178.0474.Found: 178.0481.

(1R,4S)-4-Amino-3-trifluoromethylcyclopent-2-enecarboxyl-ic Acid (6). General Procedure III. Compound24a(44 mg, 0.249mmol) was dissolved in 4 N aqueous HCl (5 mL), and the resultingclear solution was heated to 70-75 °C and stirred for 40-45 min.The resulting colorless clear solution was extracted with EtOAc (3mL × 2) and evaporated to dryness under high vacuum to give thecrude product, which was further purified via ion exchangechromatography (0.15 N aqueous HCl as eluant) to give6 as awhite solid (49 mg, 85%), mp 195.0-196.0°C. 1H NMR (D2O,400.169 MHz)δ 6.96 (1H, s), 4.69 (1H, s), 3.92 (1H, s), 2.87-2.95 (1H, m), 2.29-2.35 (1H, m).19F NMR (D2O, 376.493 MHz)δ -64.35 (3F, s).13C NMR (D2O, 125.614 MHz)δ 175.12, 142.54(q, JFC ) 4.6 Hz), 130.82 (q,JFC ) 33.7 Hz), 121.62 (q,JFC )269.7 Hz), 53.64, 48.80, 32.11. MSm/z (EI, MeOH): 196.1 (M)+,179.1 (M- OH)+, 159.1. HRMS calcd for C7H9O2NF3: 196.0580.Found: 196.0576. Anal. (C7H9O2NF3) C, H, N.

(1S,4R)-6-Pentafluoroethyl-2-(4-methoxybenzyl)-2-azabicyclo-[2.2.1]hept-5-en-3-one (22b).A 5 mL screw-capped Pyrex tubewas charged with CuI (19 mg, 0.10 mmol), KF (14.5 mg, 0.25mmol), and a stirring bar. The tube was dried with a heat gun undervacuum for 20 min and filled with argon. Compound17 (18 mg,0.05 mmol) in anhydrous NMP (1 mL) was added via syringe tothe above reaction tube followed by TMSC2F5 (48 mg, 0.25 mmol)in anhydrous DMF (1 mL). The resulting suspension was heatedto 80°C and stirred vigorously for 48 h. The reaction mixture wasthen evaporated under high vacuum, and the residue was purifiedvia column chromatography on silica gel (hexanes/EtOAc, 3:1) togive 22b (10 mg, 57%) as a light-yellow oil.1H NMR (400.168MHz, CDCl3) δ 7.17-7.19 (3H, m), 6.89 (2H, d,J ) 8.8 Hz),4.80 (1H, d,J ) 15.2 Hz), 4.26 (1H, s), 3.82 (3H, s), 3.60 (1H, s),3.52 (1H, d,J ) 14.4 Hz), 2.40 (1H, d,J ) 6.4 Hz), 2.32 (1H, d,J ) 8.0 Hz). 19F NMR (376.49 MHz, CDCl3) δ -84.12 (3F, s),-113.67 (2F, ab,Jab ) 279.4 Hz).13C NMR (CDCl3, 125.6 MHz)δ 177.59, 159.30, 144.14 (t,JCF ) 8.0 Hz), 143.03 (t,JCF ) 26.0Hz), 129.51, 128.21, 118.81 (dt,JCF ) 285.4 Hz, 37.8 Hz), 114.24,61.90, 59.79, 55.32, 54.54, 46.64. MSm/z (EI, DCM): 347.1 (M)+,121.0 (PMB)+. HRMS calcd for C16H14O2NF5: 347.0939. Found:347.0933.

(1S,4R)-6-Pentafluoroethyl-2-azabicyclo[2.2.1]hept-5-en-3-one (24b)was synthesized from22b in a yield of 74% using generalprocedure II.1H NMR (500 MHz, CDCl3) δ 7.14 (1H, s), 6.64(1H, br. s), 4.52 (1H, s), 3.43 (1H, s), 2.52 (1H, d,J ) 7.0 Hz),2.41 (1H, d,J ) 7.0 Hz).19F NMR (376 MHz, CDCl3) δ -84.45(3F, s),-116.41 (2F, ab,Jab ) 276.3 Hz,∆V ) 268.h Hz).13CNMR (CDCl3, 125.6 MHz) δ 182.28, 143.40-143.78 (m, 2C),111.80-122.15 (m, 2C, C2F5), 60.72, 59.23, 54.13. MSm/z (CI,DCM): 228.0 (M+ 1)+, 185.3 (M- CON)+, 165.3 (M- CONH- F)+. HRMS calcd for C16H14O2NF5: 347.0939. Found: 347.0933.

(1R,4S)-4-Amino-3-pentafluoroethylcyclopent-2-enecarboxy-lic acid (7) was synthesized using general procedure III from24bas a white solid in a yield of 82%, mp 187.0-188.5°C. 1H NMR(D2O, 499.511 MHz)δ 7.00 (1H, s), 4.71 (1H, d,J ) 3.5 Hz),


3.92 (1H, s), 2.85-2.91 (1H, m), 2.29-2.33 (1H, m).19F NMR(D2O, 376.493 MHz)δ -84.60 (3F, t,J ) 18.8 Hz),-113.07 (2F,ab,J ) 283.9 Hz,∆ ) 62.5 Hz).13C NMR (D2O, 125.614 MHz)δ 174.89, 145.37 (t,JFC ) 7.2 Hz), 129.63 (t,JFC ) 25.0 Hz),109.70-119.59 (2C, m), 54.61, 49.27, 32.10. MSm/z (EI,MeOH): 246.1 (M+ 1)+, 245.1 (M)+, 200.1 (M- CO2H)+, 176.1.HRMS calcd for C8H8O2NF5: 245.0470. Found: 245.0475. Anal.(C8H8O2NF5‚1.5H2O) C, H, N.

(1S,4R)-6-Bromomethylene-2-(4-methoxybenzyl)-2-azabicyclo-[2.2.1]heptan-3-one (25).(Bromomethyl)triphenylphosphoniumbromide (0.567 g, 1.3 mmol) was suspended in dry THF (4 mL),cooled to-78 °C, and then treated by addition oftBuOK (1.0 Min THF, 1.2 mL, 1.2 mmol). The resulting brown suspension wasstirred at-78 °C for 1.5 h and then treated with16 (0.246 g, 1.0mmol) in dry THF (1 mL). The resulting light-brown mixture wasallowed to gradually warm to room temperature and was stirredovernight. The reaction was quenched via addition of water, andthe mixture was extracted with dichloromethane (10 mL× 3). Thecombined organic layers were washed with brine (20 mL× 2) anddried over anhydrous MgSO4. The solvent was removed undervacuum, and the residue was purified via column chromatographyon silica gel (hexanes/EtOAc, 1:1) to give25a(Z-isomer, 100 mg,31%) as a light-yellow oil and25b (E-isomer, 131 mg, 41%) as awhite solid.

25a: 1H NMR (500 MHz, CDCl3) δ 7.26 (2H, d,J ) 8.0 Hz),6.88 (2H, d,J ) 8.0 Hz), 6.01 (1H, s), 4.76 (1H, d,J ) 15.0 Hz),4.35 (1H, s), 3.81 (3H, s), 3.70 (1H, d,J ) 15.0 Hz), 3.02 (1H, s),2.47 (1H, d,J ) 16.0 Hz), 2.31 (1H, d,J ) 15.5 Hz), 1.97 (1H, d,J ) 9.5 Hz), 1.54 (1H, d,J ) 10.0 Hz). 13C NMR (126 MHz,CDCl3) δ 177.51, 159.15, 143.88, 129.64, 128.94, 114.01, 97.82,61.88, 55.31, 45.81, 44.36, 39.78, 32.77. MSm/z (ESI, MeOH):344.6 (M + Na)+, 346.5 (M + 2 + Na)+. HRMS calcd forC15H16O2NBr: 321.0359. Found: 321.0359.

25b: 1H NMR (500 MHz, CDCl3) δ 7.14 (2H, d,J ) 8.5 Hz),6.86 (2H, d,J ) 8.5 Hz), 6.10 (1H, s), 4.62 (1H, d,J ) 15.0 Hz),3.86 (1H, s), 3.80 (3H, s), 3.79 (1H, d,J ) 15.0 Hz), 2.97 (1H, s),2.43 (1H, dt,J ) 16.5 Hz, 3.0 Hz), 2.26 (1H, d,J ) 16.5 Hz),2.06 (1H, d,J ) 9.5 Hz), 1.60 (1H, d,J ) 9.5 Hz).13C NMR (126MHz, CDCl3) δ 177.79, 159.19, 144.28, 129.42, 128.53, 114.15,100.00, 63.46, 55.33, 44.89, 43.95, 41.16, 33.49. MSm/z (ESI,MeOH): 344.6 (M+ Na)+, 346.2 (M+ 2 + Na)+. HRMS calcdfor C15H16O2NBr: 321.0359. Found: 321.0359.

(Z)-(1S,4R)-2-(4-Methoxybenzyl)-6-(2,2,2-trifluoroethylidene)-2-azabicyclo[2.2.1]heptan-3-one (26a)was synthesized in a 95%yield from 25ausing general procedure I (reaction time, 20 h).1HNMR (400 MHz, CDCl3) δ 7.18 (2H, d,J ) 8.8 Hz), 6.88 (2H, d,J ) 8.4 Hz), 5.68 (1H, q,J ) 8.0 Hz), 4.81 (1H, d,J ) 14.8 Hz),4.44 (1H, s), 3.82 (1H, s), 3.58 (1H, d,J ) 15.2 Hz), 2.95 (1H, s),2.57 (1H, d,J ) 17.2 Hz), 2.41 (1H, d,J ) 17.2 Hz), 2.06 (1H, d,J ) 8.8 Hz), 1.58 (1H, d,J ) 10.4 Hz).19F NMR (376.49 MHz,CDCl3) δ -58.02 (3F, m). MSm/z (EI, DCM): 311.1 (M)+, 312.1(M + 1)+, 121.0 (PMB)+. HRMS calcd for C16H16O2NF3: 311.1128.Found: 311.1133.

(E)-(1S,4R)-2-(4-Methoxybenzyl)-6-(2,2,2-trifluoroethylidene)-2-azabicyclo[2.2.1]heptan-3-one (26b)was synthesized in 95%yield from 25b using general procedure I (reaction time, 20 h).1HNMR (400 MHz, CDCl3) δ 7.15 (2H, d,J ) 8.8 Hz), 6.87 (2H, d,J ) 8.4 Hz), 5.51 (1H, q,J ) 7.2 Hz), 4.57 (1H, d,J ) 14.4 Hz),3.90 (1H, d,J ) 15.2 Hz), 3.86 (1H, s), 3.80 (3H, s), 2.98 (1H, s),2.62 (1H, dq,J ) 17.2 Hz, 2.4 Hz), 2.48 (1H, dq,J ) 17.2 Hz, 2.4Hz), 2.05 (1H, d,J ) 9.2 Hz), 1.56 (1H, d,J ) 10.0 Hz). 19FNMR (376.49 MHz, CDCl3) δ -60.98 (3F, m). MSm/z (EI,DCM): 311.1 (M)+, 312.1 (M+ 1)+, 121.0 (PMB)+. HRMS calcdfor C16H16O2NF3: 311.1128. Found: 311.1133.

(Z)-(1S,4R)-6-(2,2,2-Trifluoroethylidene)-2-azabicyclo[2.2.1]-heptan-3-one (27a)was synthesized in 74% yield from26ausinggeneral procedure II.1H NMR (400 MHz, CDCl3) δ 6.30 (1H, s),5.58 (1H, q,J ) 8.0 Hz), 4.61 (1H, s), 2.83 (1H, s), 2.55 (1H, d,J ) 17.2 Hz), 2.36 (1H, d,J ) 16.4 Hz), 2.18 (1H, dd,J ) 10.4Hz, 1.2 Hz), 1.63 (1H, d,J ) 9.6 Hz).19F NMR (376 MHz, CDCl3)δ -58.28 (3F, m).13C NMR (CDCl3, 125.6 MHz)δ 179.92, 152.44

(q, JFC ) 5.5 Hz), 122.93 (q,JFC ) 270.1 Hz), 110.02 (q,JFC )34.8 Hz), 56.67, 43.58, 41.49, 32.23. MSm/z (EI, DCM): 192 (M+ 1)+, 191 (M)+, 79.1. HRMS calcd for C8H8ONF3: 191.0553.Found: 191.0559.

(E)-(1S,4R)-6-(2,2,2-Trifluoroethylidene)-2-azabicyclo[2.2.1]-heptan-3-one (27b)was synthesized in a 74% yield from26businggeneral procedure II.1H NMR (400 MHz, CDCl3) δ 6.57 (1H, s),5.77 (1H, d,J ) 7.6 Hz), 4.13 (1H, s), 2.87 (1H, s), 2.65 (1H, d,J ) 16.0 Hz), 2.48 (1H, d,J ) 16.8 Hz), 2.14 (1H, d,J ) 9.6 Hz),1.62 (1H, d,J ) 9.6 Hz).19F NMR (376 MHz, CDCl3) δ -61.08(3F, m).13C NMR (CDCl3, 125.6 MHz)δ 180.27, 151.69 (q,JFC

) 4.6 Hz), 123.20 (q,JFC ) 270.1 Hz), 110.82 (q,JFC ) 35.7 Hz),60.10, 44.60, 41.52, 30.53. MSm/z (EI, DCM): 192 (M + 1)+,191 (M)+, 163 (M - CO)+, 79. HRMS calcd for C8H8ONF3:191.0553. Found: 191.0553.

(1S,3S)-(Z)-3-Amino-4-(2,2,2-trifluoroethylidene)cyclopen-tanecarboxylic acid (8)was synthesized in a 66% yield as a whitesolid from 27a using general procedure III; mp 212.0-214.0°C.1H NMR (D2O, 499.511 MHz)δ 5.97 (1H, q,J ) 7.5 Hz), 4.37(1H, s), 3.09-3.16 (2H, m), 2.92-2.95 (1H, m), 2.56-2.62 (1H,m), 1.98 (1H, q,J ) 12.5 Hz).19F NMR (D2O, 376.493 MHz)δ-60.78 (3F, d,JHF ) 6.0 Hz). 13C NMR (D2O, 125.614 MHz)δ177.93, 150.92 (q,JFC ) 5.0 Hz), 123.11 (q,JFC ) 270.2 Hz),114.77 (q,JFC ) 35.3 Hz), 53.76, 40.69, 32.95, 32.43. MSm/z(EI, MeOH): 209.1 (M)+, 208.1 (M - 1)+, 189.1 (M - H2O -2)+, 164.1 (M- CO2H)+. HRMS calcd for C8H10O2NF3: 209.0658.Found: 277.0656. Anal. (C8H10O2NF3‚0.25H2O) C, H, N.

(1S,3S)-(Z)-3-Amino-4-(2,2,2-trifluoroethylidene)cyclopen-tanecarboxylic acid (9)was synthesized from27b in a 79% yieldas a white solid using general procedure III, mp 206.0-207.5°C.1H NMR (D2O, 499.749 MHz)δ 6.09 (1H, qd,J ) 9.0 Hz, 1.5Hz), 4.65 (1H, s), 3.02-3.09 (1H, m), 2.91-2.92 (2H, m), 2.57-2.63 (1H, m), 2.13-2.18 (1H, m).19F NMR (D2O, 376.493 MHz)δ -60.37 (3F, d,JHF ) 7.9 Hz).13C NMR (D2O, 125.673 MHz)δ178.64, 149.42 (q,JFC ) 5.4 Hz), 120.43 (q,JFC ) 269.3 Hz),116.73 (q,JFC ) 34.3 Hz), 50.15, 40.74, 37.00, 34.44. MSm/z(EI, MeOH): 210.1 (M+ 1)+, 209.1 (M)+, 191.1 (M - H2O)+,164.1 (M - CO2H)+. HRMS calcd for C8H10O2NF3: 209.0664.Found: 277.0675. Anal. (C8H10O2NF3‚0.25H2O) C, H, N.

(1S,4R)-6-Dibromomethylene-2-(4-methoxybenzyl)-2-azabi-cyclo[2.2.1]heptan-3-one (28).Compound16 (0.246 g, 1.0 mmol),carbon tetrabromide (0.498 g, 1.5 mmol), and triphenylphosphine(0.787 g, 3.0 mmol) were dissolved in anhydrous toluene (10 mL),and the resulting light-yellow suspension was stirred while refluxingunder argon for 22 h. The reaction mixture was then filtered, washedwith benzene, and evaporated to give the crude product, which waspurified via column chromatography on silica gel (hexanes/EtOAc,1:1) to give28 (377 mg, 94%) as a light-yellow oil.1H NMR (500MHz, CDCl3) δ 7.21 (2H, m), 6.85 (2H, m), 4.72 (1H, d,J ) 12.5Hz), 4.20 (1H, s), 3.73-3.78 (4H, m), 3.03 (1H, s), 2.45 (1H, d,J) 16.5 Hz), 2.29 (1H, d,J ) 16.5 Hz), 2.02 (1H, s), 1.63 (1H, s).13C NMR (126 MHz, CDCl3) δ 177.41, 159.15, 145.83, 129.45,128.51, 114.04, 81.58, 64.27, 55.28, 45.66, 44.49, 40.84, 36.61.MS m/z (ESI, MeOH): 399.9 (M)+, 401.9 (M+ 2)+, 404 (M +4)+, 322.0 (M- Br)+, 293.9 (M- CH3OC6H4)+. HRMS calcd forC15H15O2NBr2: 398.9464. Found: 398.9460.

(1S,4R)-2-(4-Methoxybenzyl)-6-(2,2,2-trifluoro-1-trifluoro-methylethylidene)-2-azabicyclo[2.2.1]heptan-3-one (29)was syn-thesized from28 in an 82% yield using general procedure I (50 h).1H NMR (400 MHz, CDCl3) δ 7.15 (2H, d,J ) 8.8 Hz), 6.88 (2H,d, J ) 8.8 Hz), 4.82 (1H, d,J ) 15.2 Hz), 4.62 (1H, s), 3.81 (3H,s), 3.65 (1H, d,J ) 15.2 Hz), 3.03 (1H, s), 2.82 (1H, d,J )18.4 Hz), 2.73 (1H, d,J ) 18.4 Hz), 2.12 (1H, d,J ) 10.4 Hz),1.63 (1H, d,J ) 10.4 Hz). 19F NMR (376.49 MHz, CDCl3) δ-57.12 to-57.03 (3F, m),-59.97 to-59.91 (3F, m). MSm/z(EI, DCM): 379.1 (M)+, 380.1 (M+ 1)+, 136.1 (PMB+ NH)+,121.0 (PMB)+. HRMS calcd for C16H16O2NF3: 379.1001. Found:379.1003.

(1S,4R)-6-(2,2,2-Trifluoro-1-trifluoromethylethylidene)-2-aza-bicyclo[2.2.1]heptan-3-one (30)was synthesized from29 in a 56%yield using general procedure II.1H NMR (400 MHz, CDCl3) δ


5.62 (1H, s), 4.78 (1H, s), 2.96 (1H, s), 2.83 (1H, s), 2.74 (1H, s),2.29 (1H, s), 1.71 (1H, s).19F NMR (376 MHz, CDCl3) δ -57.32(3F, s),-60.25 (3F, s). MSm/z (EI, DCM): 260.1 (M+ 1)+, 259.1(M)+, 189.1 (M- CF3)+. HRMS calcd for C9H7ONF6: 259.0426.Found: 259.0434.

(1S,3S)-3-Amino-4-(2,2,2-trifluoro-1-trifluoromethylethylidene)-cyclopentanecarboxylic acid (10)was synthesized from30 in a77% yield as a white solid using general procedure III; mp 198.0-199.5 °C. 1H NMR (D2O, 400.169 MHz)δ 4.92 (1H, s), 3.29-3.34 (1H, m), 3.16-3.20 (2H, m), 2.57-2.65 (1H, m), 2.20-2.27(1H, m).19F NMR (D2O, 376.493 MHz)δ -59.97 (3F, q,J ) 9.0Hz), -60.47 (3F, q,J ) 9.0 Hz).13C NMR (D2O, 100.749 MHz)δ 178.08, 158.14, 122.40, 119.16-119.67 (m), 52.56 (d,JFC ) 2.3Hz), 40.73, 36.31 (q,JFC ) 3.8 Hz), 33.68. MSm/z (EI, MeOH):278.1 (M + 1)+, 277.1 (M)+, 259.1 (M - H2O)+, 232.0 (M -CO2H)+. HRMS calcd for C9H9O2NF6: 277.0538. Found: 277.0545.Anal. (C9H9O2NF6‚H2O) C, H, N.

Enzyme and Assays.GABA aminotransferase was isolated frompig brains by a known procedure.34 Succinic semialdehyde dehy-drogenase (SSDH) was isolated from GABAse, a commerciallyavailable mixture of SSDH and GABA aminotransferase, byinactivation of the GABA aminotransferase with gabaculine asdescribed previously.35 GABA activity assays were carried out usinga modification of the coupled assay developed by Scott andJakoby.36 The assay solution contains 11 mM GABA, 5.3 mMR-ketoglutarate, 1.1 mM NADP+, and 5 mMâ-mercaptoethanolin 100 mM potassium pyrophosphate, pH 8.5, and excess SSDH.By use of this assay, the change in absorbance at 340 nm indicatesproduction of NADPH, which is directly proportional to the activityof GABA aminotransferase.

Time-Dependent Inactivation of GABA Aminotransferase.Incubation solutions (200µL) contained enzyme (30µL), potassiumpyrophosphate buffer (volume dependent on volume of inhibitor,50 mM, pH 8.5), R-ketoglutarate (20µL, 16 mM in 50 mMpotassium pyrophosphate buffer, pH 8.5), 2-mercaptoethanol (2.0mM), and various volumes of the inhibitor solutions (6, 8, and9,0.2 M in doubly distilled H2O). The concentration of inhibitorswas calculated on the basis of a total volume of 200µL. At timeintervals, aliquots (27µL) from the incubation solution were addedto the assay solution (568µL) with excess succinic semialdehydedehydrogenase (SSDH). Rates were measured spectrophotometri-cally at 340 nm, and the logarithm of the remaining activity(percentage) was plotted against time for each concentration todetermine the half-time. Then a secondary plot of half-time versusthe reciprocal of the inhibitor concentration was obtained todetermine thekinact andKI values.

Exhaustive Dialysis of 6, 8, and 9 Inactivated GABA-AT withPotassium Pyrophosphate Buffer.Incubation solutions (200µL)contained enzyme (30µL), potassium pyrophosphate buffer (147µL, 50 mM, pH 8.5),R-ketoglutarate (20µL, 16 mM in 50 mMpotassium pyrophosphate buffer, pH 8.5), 2-mercaptoethanol (2.0mM), and the inhibitor solutions (6, 8, and9, 3.0 µL, 0.2 M indoubly distilled H2O). A control solution (200µL) containingenzyme (30µL), potassium pyrophosphate buffer (150µL, 50 mM,pH 8.5), R-ketoglutarate (20µL, 16 mM in 50 mM potassiumpyrophosphate buffer, pH 8.5), 2-mercaptoethanol (2.0 mM), andno inhibitor solution was also prepared. The incubation solutionwas allowed to stand at room temperature for about 1 h toensurethat most of the enzyme was inactivated. The incubation solutionsand the control solution were injected into a Slide-A-Lyzer dialysiscassette (extra strength, 10000 MWCO, 0.5-3.0 mL capacity),respectively, and stirred in a 1 L beaker containing a stirring barand 800 mL of 50 mM potassium pyrophosphate buffer (pH 8.5).The buffer was changed every 4 h, at which point an aliquot wastaken out to assay.

Substrate Protection against Inhibition of GABA-AT byCompounds 6, 8, and 9.Incubation solutions (200µL) containedenzyme (30µL), potassium pyrophosphate buffer (volumes de-pendent on the volume of substrate, 50 mM, pH 8.5),R-ketoglu-tarate (20µL, 16 mM in 50 mM potassium pyrophosphate buffer,pH 8.5), 2-mercaptoethanol (2.0 mM), inhibitor solutions (2µL or

0 µL in control, 6, 8, or 9 in doubly distilled H2O, 0.2 M), andGABA solution (0µL (control), 17.6 and 35.2µL). The concentra-tions of GABA in the incubation solutions were 0 (control), 3, and6 mM, respectively. At time intervals, aliquots (27µL) from theincubation solution were added to the assay solution (568µL) withexcess succinic semialdehyde dehydrogenase (SSDH). Rates weremeasured spectrophotometrically at 340 nm, and the remainingactivity (percentage) was determined against the control and plottedversus time to determine thet1/2.

Fluoride Ion Released During Inhibition of GABA-AT by5-10. GABA-AT (25 µL) was incubated for 3 h with each of thesix compounds5-10 (0.8 mM) in 50 mM potassium pyrophosphatebuffer, pH 8.5, containing 2.0 mMâ-mercaptoethanol and 0.64 mMR-ketoglutarate in a total volume of 250µL at 25 °C. A controlcontaining no enzyme (25µL of buffer was added instead) wasalso run. The fluoride ion concentration (200µL) of each samplemixed with 1.00 mL of total ionic strength adjusting buffer (TISABlow level), 790µL of potassium pyrophosphate buffer, and 10µLof diluted standard fluoride solution (2.38× 10-4 M), was measuredwith a fluoride ion electrode.

Fluoride Ion Release during Inhibition of GABA-AT by 6and 7 with Time. GABA-AT (120 µL) was incubated with6 or 7(12 µL, 0.2 M in doubly distilled water) in 50 mM potassiumpyrophosphate buffer, pH 8.5, containing 2.0 mMâ-mercapto-ethanol and 0.64 mMR-ketoglutarate in a total volume of 1.2 mLat 25°C. Another two samples containing no GABA-AT but serumalbumin or just potassium pyrophosphate buffer (control) were alsorun. At time intervals, 200µL of each sample was removed andmixed with 1.00 mL of total ionic strength adjusting buffer (TISABlow level), 790µL of potassium pyrophosphate buffer, and 10µLof diluted standard fluoride solution (2.38× 10-4 M). The fluorideion concentration was measured with a fluoride ion electrode.

Acknowledgment. The authors are grateful to the NationalInstitutes of Health (Grant GM66132) for financial support ofthis research.

Supporting Information Available: Results from combustionanalyses. This material is available free of charge via the Internetat http://pubs.acs.org.

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JM0608715


fluorinated conformationally restricted γ-aminobutyric acid aminotransferase inhibitors

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